Abstract A poorly evolved area of infectious disease research includes fully understanding the life cycle of a pathogen within different natural environments. Tick-borne relapsing fever (RF) spirochetes are one such pathogen that have adapted to colonize ticks and lice. The Ornithdoros tick vector is long lived (10 ? 20 years) and maintain the pathogens throughout their life history, with populations of spirochetes colonizing the midgut and salivary glands. These ticks are rapid feeders, completing the blood meal within 5 ? 60 minutes of attachment. Thus, the population of spirochetes in the salivary gland enters the mammalian host to continue the pathogen's life cycle. Since there is insufficient time for midgut population to reach the salivary glands during the short bloodmeal, the role of this population in the pathogens life cycle is unclear. Additionally, the genetic constituents expressed by spirochetes in the midgut and salivary glands are virtually unknown. With the longevity of the tick vector and their ability to continuously transmit RF spirochetes over their life time, one aspect of pathogenesis relevant to public health is understanding pathogen replication in the vector and identifying molecular events that RF spirochete utilize for vector colonization and transmission. By developing the Borrelia turicatae ? Ornithodoros turicata model we can now define the pathogen's life cycle and identify genetic targets that are likely essential in the transmission cycle. This study will test the hypothesis that B. turicatae colonizing the midgut of infected ticks replicate following a bloodmeal and replenishes the salivary glands, and there are specific gene subsets expressed by the bacteria in each tissue. The following two Specific Aims are proposed: Aim 1: Define the life cycle of B. turicatae in the tick by assessing spirochete replication in the midgut before and at time points after the bloodmeal. These results will indicate whether RF spirochetes multiply in the tick, and implicate the midgut as the site of replication. Aim 2: Delineate expression patterns of genes localized on the B. turicatae 150 kb linear megaplasmid (lp150) in the tick midgut and salivary glands. Previous studies suggest that lp150 is likely essential in the life cycle of RF spirochetes, and this plasmid's genes will be assessed to determine their expression in the tick midgut and salivary glands. Determining the importance of RF spirochete replication in the tick, and identifying genes expressed by the pathogens in the midgut and salivary glands will guide strategies to disrupt the bacteria's life cycle. The results will focus future research efforts to neutralize midgut population from replicating, and prevent the salivary gland population from establishing mammalian infection. These findings will be broadly applicable to Old and New World pathogens, and address NIAID's mission to better understand and prevent disease.